The principal object of this invention is to provide an improved and simplified method for extracting protease-inhibitors from animal tissues which contain such inhibitors.
Another object of this invention is to provide a method for extracting protease inhibitors which contain a very reduced percentage of pyrogenic substances and polyanionic pollutants such as mucopolysaccharides and nucleic acids.
Still another object of the present invention is to provide a method for extracting protease inhibitors from animal tissues which contain such inhibitors without interfering with other methods directed towards the extraction of other useful substances, such as heparin, from animal tissues; it will be appreciated that the method of the present invention permits to extract heparin, if so desired, from an intermediate fraction which contains heparin, it being however understood that the method according to this invention is directed essentially and predominantly to the extraction of protease inhibitors.
According to the preferred embodiment of this invention, a method is suggested for extracting a protease-inhibitor from protease-inhibitor-containing fresh or frozen organs of slaughtered animals, said organs showing no signs of incipient or progressed azymic autolysis, said method comprising the steps of
(a) comminuting said organs;
(b) subjecting said comminuted organs to enzymolysis by contacting them, within an aqueous medium, with at least one proteolytic enzyme selected from the group of the proteolytic enzymes which do not naturally occur in said organs and which are inactive towards said protease-inhibitor to be extracted;
(c) discontinuing said enzymolysis and collecting a lysate aqueous solution by subjecting the aqueous medium containing said comminuted organs and said at least one proteolytic enzyme to filtration;
(d) adding a quaternary ammonium base to said lysate aqueous solution to obtain the precipitation of an insoluble fraction;
(e) filtering off said insoluble fraction and collecting the inhibitor-containing filtrate, and
(f) recovering said protease-inhibitor from said filtrate.
It is preferred that the proteolytic enzyme referred to above be a member selected from the group consisting of papain, ficin bromelin, Alkalase (R.T.M.) and Neutrase (R.T.M.).
The enzymolysis is stopped, at the proper instant of time, by any of the method as conventionally used for such an operation: heating to 90.degree. C. and over is a preferred method, but other methods can be used, such as the addition of a deproteinating agent or otherwise, since it matters to stop the enzymolysis when desired, rather than the means used for the discontinuation.
The enzymolysis should be conducted under the optimum conditions for the proteolytic enzyme selected. If papain is used, a temperature of 60.degree. C..+-.2.degree. C. with a pH from 5.0 to 6.0 are preferred conditions, and a pH of 5.5 is an optimum. If ficin is the selected proteolytic enzyme, the conditions preferred for papain apply again. If bromelin is used, a lower temperature, such as 37.degree. C..+-.2.degree. C. is preferred, the preferential pH being near 6.
If the proteolytic enzyme used is Alkalase (R.T.M.) the optimum temperature is 65.degree. C..+-.2.degree. C., the pH is preferred to be near 7.2 and the enzymolysis time does not exceed 150 mins. When using Neutrase (R.T.M.) the preferred temperature is 60.degree. C..+-.2.degree. C., the preferred pH in the neighbourhood of 6.7 and the time, again, does not exceed 150 mins. for the enzymolysis step.
According to the invention, the preferred quaternary ammonium bases are cetylipiridinium and cetyltrimethylammonium bromide.
Other features, properties and advantages of the invention will become apparent as the present description proceeds.